Most viruses that replicate in the cytoplasm of host cells form neoorganelles that serve as sites of viral genome replication and particle assembly. These highly specialized inclusion structures concentrate viral replication proteins and nucleic acids, prevent activation of cell-intrinsic defenses, and coordinate release of progeny particles. Despite the importance of inclusion complexes in viral replication, there are key gaps in knowledge about how these organelles form and mediate their functions. The proposed research uses reovirus, a genetically tractable virus that has been linked to celiac disease and shows promise for oncolytic applications, to elucidate mechanisms of double-stranded (ds) RNA virus inclusion formation, genome replication, and progeny particle assembly and release. Like other dsRNA viruses, which include important pathogens of animals (orbiviruses) and humans (rotaviruses), reovirus inclusions are nucleated by viral nonstructural proteins that recruit viral structural proteins for genome replication and particle assembly. We have discovered that (i) reovirus inclusions originate from the endoplasmic reticulum (ER), (ii) capsid assembly requires the TRiC chaperonin, and (iii) progeny particles are transported from inclusions and released using a vesicular sorting mechanism dependent on modified lysosomes and actin. Three integrated specific aims are proposed to fill key knowledge gaps about reovirus inclusion biogenesis, capsid assembly, and particle egress. In Specific Aim 1, functions of reovirus nonstructural proteins ?NS and NS in inclusion formation and genome synthesis will be elucidated using biochemical and cell-imaging approaches. The structure of ?NS will be determined to facilitate analysis of its function in ER reorganization and dsRNA synthesis. In Specific Aim 2, interactions between TRiC and reovirus outer-capsid protein ?3 will be defined using mass spectrometry and cryo-EM analysis. The function of the TRiC-loading protein, prefoldin, in ?3 maturation will be defined using in vitro protein-folding assays. The protein-folding network required to assemble ?3 onto newly formed virions will be defined using gene-targeting and biochemical assays. In Specific Aim 3, the lipid-biosynthetic and transport pathways required for reovirus egress will be elucidated by defining a function for Niemann-Pick disease type C1 protein, identified in RNA interference and CRISPR screens, in the egress process. The mechanism by which mature virions are transferred into membranous, multilamellar structures and then to smaller membranous carriers to reach the plasma membrane will be defined using light and electron microscopy. The function of actin and myosin 9 in inclusion biogenesis, particle assembly, and viral egress will be defined using pharmacologic inhibitors and cell imaging. These studies will enhance an understanding of mechanisms by which pathogenic viruses alter cellular architecture to engineer inclusion organelles, assemble progeny particles, and exit infected cells. We anticipate that this information will foster development of antiviral drugs that impede these essential viral replication steps and enhance the use of reovirus as an oncolytic therapeutic.